A Comprehensive Comparison of Rapid RNA Extraction Methods for Detection of SARS-CoV-2 as the Infectious Agent of the Upper Respiratory Tract using Direct RT-LAMP Assay

Abstract

Background: The current COVID-19 pandemic has highlighted the need for faster and more cost-effective diagnostic methods. The RNA extraction step in current diagnostic methods, such as real-time qPCR, increases the cost and time required for testing. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is a promising technique for developing diagnostic tests with desired sensitivity and specificity without the need for RNA extraction. Materials and Methods: An RT-LAMP assay was developed to detect SARS-CoV-2 with a sensitivity of 0.5 copies of positive control plasmid per microliter in 40 min. Several rapid RNA extraction protocols were evaluated using different reagents, including bovine serum albumin, Triton X-100, Tween 20, proteinase K, guanidine hydrochloride, guanidinium isothiocyanate (GITC), and thermal treatment. Finally, the sensitivity and specificity of the developed direct RT-LAMP were determined using 150 upper respiratory tract samples. Results: Method 10 was selected as the most efficient protocol for the RNA extraction step. The sensitivity and specificity of the developed direct RT-LAMP assay with clinical samples were estimated at 98.4% and 88.8%, respectively. Conclusion: These results suggest that the combination of GITC and Triton X-100 detergent is a highly efficient method for RNA extraction and direct RT-LAMP detection of SARS-CoV-2 in clinical samples, providing a valuable tool for the rapid and cost-effective diagnosis of COVID-19.

Keywords

• rapid diagnostic tests
• rt-lamp assay
• sars-cov-2